The overall goal of this project is to elucidate the cellular mechanisms by which the PDZ scaffolding protein NHERF1 regulates parathyroid hormone receptor (PTHR)-mediated signaling and function in bone. Mice with targeted deletion of NHERF1 exhibit a bone phenotype including an increased fracture rate, as do patients with NHERF1 mutations. We showed that these effects are a direct consequence of the absence or diminished abundance of NHERF1 in osteoblasts. It is not known how NHERF1 expression is regulated or how its conformations affect biological function. NHERF1 can assume open and closed conformations but it is unknown how these conformations are regulated or how they affect biological function. We demonstrate that vitamin D upregulates NHERF1 mRNA and protein in primary osteoblasts. Such an action could help explain the beneficial skeletal effects of vitamin D. Further, NHERF1 is a phospho-protein that assembles a macromolecular complex with the PTHR and ezrin. PTH-induced NHERF1 phosphorylation dissembles the PTHR-NHERF1-ezrin complex permitting PTHR endocytosis and termination of PTH action. The unifying hypothesis guiding this proposal is that the dual events of vitamin D stimulation of NHERF1 expression and of PTH-induced phosphorylation regulate the assembly and disassembly of functional NHERF1 macromolecular complexes. Three specific aims are developed to assess this idea. Aim 1 tests the hypothesis that vitamin D upregulates NHERF1 and defines the gene locus of this action; Aim 2 explores PTH-regulated NHERF1 phosphorylation and associated conformations; Aim 3 analyzes the proposition that by upregulating NHERF1 expression in intact mice, vitamin D increases membrane-delimited PTHR, thereby augmenting the effect of PTH on mineralization and bone growth. Biophysical, genetic, biochemical, and structural biological approaches will be applied to characterize these effects. Innovative chemical biological and genetic experiments along with high-content modeling will be performed. The outcomes will reveal new details of the regulation of osteoblast function and the role of NHERF1 in normal bone remodeling and in mineral bone disorder associated with chronic kidney disease (CKD-MBD). The results will help define potential therapeutic targets for improved treatment of osteoporosis and other metabolic bone diseases.